1. Field
The present invention relates to a novel use of an EC-SOD protein and a method for preparing thereof. More particularly, the present invention relates to compositions for preventing or treating angiogenesis-mediated disease or allergic diseases which contain, an EC-SOD protein or a vector having a polynucleotide encoding said EC-SOD proteins an active ingredient.
2. Discussion of the Background
Superoxide dismutases (SODs) function to protect cells by removing reactive oxygen species and enabling other antioxidant enzymes, and SODs known to date include Cu/Zn SOD (SOD 1) containing copper and zinc atoms, Mn SOD (SOD 2) containing a manganese atom, and extracellular superoxide dismutase (EC-SOD) present on the cell surface or in the extracellular fluid.
Particularly, EC-SOD contains copper and zinc atoms as in Cu/Zn SOD, but is characterized in that a heparin binding domain is present in the C-terminal end. Since EC-SOD has the heparin-binding domain, it is assumed that EC-SOD will function to protect cell membranes by binding to the cell membranes. According to literatures, it was known that EC-SOD plays a role in the body's defense mechanism in serums and extracellular matrices (Marklund et al, Biochem. J. 266, 213-219, 1990; Su et al., Am J Respir Cell Mol Biol., February 16(2), 162-70, 1997; et al., Thromb. Vasc. Bio. 18, 157-167, 1998). In addition, it was reported that the heparin-binding domain of EC-'SOD acts as a nuclear localization signal, so that it is located within the nuclei of thymus and testis cells so as to protect genomic DNA from oxidative stress and to regulate the DNA transcription sensitive to oxidation-reduction reaction (Ookawara T et al., BBRC, 296, 54-61, 2002). The present inventors reported in Korean Patent Registration No. 10-0676502 that EC-SOD has the effect of treating skin diseases such as psoriasis by removing reactive oxygen species in skin cells and inhibiting the over-proliferation of epidermal cells.
Meanwhile, angiogenesis is the process by which new capillary blood vessels are formed from preexisting vessels. Angiogenesis normally occurs only in some specific situations, including embryonic development, wound healing and the female reproductive cycle, and does not substantially occur in normal conditions. However, when angiogenesis is not regulated correctly, diseases such as cancer may be induced.
The angiogenic process consists of several steps that include stimulation of endothelial cell growth by tumor cytokine, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), degradation of extracellular matrix proteins by matrix metalloprotease (MMP), and migration of endothelial cells, which is mediated by membrane adhesion molecules, endothelial cell differentiation and tube formation (Bussolino, F. et al., Trends Biochem. Sci. 22:251-256, 1997; Kuwano, M. et al., Intern. Med. 40:565-572, 2001; Risau, W. Angiogenesis and endothelial cell function. Arzneimittelforschung 44:416-417, 1994). Thus, inhibition of these above processes has been proposed as a new therapeutic strategy for the treatment of angiogenesis-mediated diseases, including cancer and other human diseases. For this purpose, a variety of angiogenesis inhibitors have been developed. The inhibitors, which are natural or synthetic, include protease inhibitors, tyrosine kinase inhibitors, chemokines, interleukins, and proteolytic fragments of matrix proteins (Abedi, H. et. al., J. Biol. Chem. 272:15442-15451, 1997; Cao, Y., Int. J. Biochem. Cell Biol. 33:357-369, 2001; Fong, T. A et al., Cancer Res. 59:99-106, 1999; Kwon, H. J. et al., Acalycigorgia inermis. J. Microbiol. Biotechnol. 11:656-662, 2001). These antiangiogenic molecules affect various processes, including the inhibition of endothelial cell proliferation, migration, protease activity, and tubule formation, as well as the induction of apoptosis (Folkman, J. et. al., Semin. Cancer Biol. 3:89-96, 1992; Kishi, K. et al., Nippon Rinsho 58:1747-1762, 2000; Marme, D., Onkologie 1:1-5, 2001). The antiangiogenic function of many of these molecules has been well studied in vitro and in vivo, and some antiangiogenic drugs are currently being tested in clinical trials (Deplanque, G. et. al., Eur. J. Cancer 36:1713-1724, 2000; Liekens, et. al., Biochem. Pharmacol. 61:253-270, 2001; Mross, K., Drug Resist. Updat. 3: 223-235, 2000).
Reactive oxygen species have been reported to be major regulators of angiogenesis (Jolanta Grzenkowicz-Wydra, et. al., Mol Cell Biochem., 264(1-2):169-81, 2004), but the relationship between SODs, which remove reactive oxygen species, and angiogenesis, has not yet been sufficiently examined. It was recently reported that the overexpression of Cu/Zn SOD (SOD 1), having copper and zinc atoms, among SODs, leads to the stimulation of angiogenesis, and thus the inhibition of SOD1 leads to the inhibition of angiogenesis (Jolanta Grzenkowicz-Wydra, et. al., Mol Cell Biochem., 264(1-2):169-81, 2004). In addition, it was reported that ATN-224, which is an inhibitor of SOD1, inhibits angiogenesis, and thus can be used as an anticancer agent, and it is in a phase II trial (Juarez et al. Clin Cancer Res., 12: 4974-4982, 2006). However, there is no report on the relationship between EC-SOD and angiogenesis.
Meanwhile, with the development of industry, allergy-causing factors have increased due to environmental pollution, an increase in new synthetic substances and a change in residential environments, and thus the number of people suffering from asthma and allergies has gradually increased. The function of the body to show specific resistance against foreign matter, which has invaded the body, is referred to as immunity. An allergy is a hypersensitive immune reaction, and typical allergic diseases include atopic dermatitis, bronchial asthma and pollenosis. Clinical symptoms resulting from allergic reactions are broadly classified into specific immune reactions at an early stage, and inflammatory reactions at a late stage. These immune reactions are mostly mediated by mast cells, which are known to be widely distributed in the systemic organs, including the skin, the respiratory organ, the gastrointestinal mucosa, the brain, around lymphatic vessels and around blood vessels and to cause allergic reactions. Mediators known to activate mast cells include an IgE antibody (FcεRI) bound to a high-affinity immunoglobulin E (IgE) receptor (FcεRI) located on the cellular membrane, compound 48/80 and the like. The activation mechanism of mast cells by IgE antibody is as follows.
When an IgE antibody bound to an IgE receptor forms a bridge with an antigen, substances, such as histamine, chondroitin sulfate, heparin and protease, stored in the mast cell granules, are released through the action of phospholipase C, protein phosphatase C and calcium ions, thus mediating the early reaction stage. Among the chemical substances, including histamine, cause clinical symptoms occurring in the early allergic reaction, histamine accounts for the largest amount. In other words, in the early allergic reaction, the expression of histamine is predominant, and clinical symptoms resulting therefrom include vasodilation, edema and the like.
Currently, antihistamine drugs or steroidal drugs are frequently used to treat allergies. However, these drugs mostly have temporary effects, and in many cases, cause serious side effects. Accordingly, there is an urgent need to develop a novel substance, which has the effect of preventing and treating rapidly increasing allergic diseases, and at the same time, has reduced side effects and lasting effects. However, whether EC-SOD has the effect of treating allergic diseases is not yet known.
Also, even if the EC-SOD has various activities, when a method for producing the EC-SOD in large amounts is not developed, there is a problem in that the EC-SOD cannot be efficiently used in industry. However, mass-production methods for proteins, developed to date, comprise transforming host cells, such as E. coli cells, with expression vectors, and inducing the expression of proteins in the transformed cells, and the mass production of proteins has problems in that it often causes the formation of inclusion bodies, and thus the original activities of the proteins are eliminated or reduced.
The present inventors have conducted studies on the EC-SOD protein, and as a result, have found that the EC-SOD shows an activity of inhibiting diseases caused by angiogenesis or allergic diseases. On the basis of this finding, the present inventors have developed a method for preparing the EC-SOD protein having the said activity, thereby completing the present invention.